5H-Cyclopenta[h]quinazoline, 6,6a,7,8,9,9a-hexahydro-7,7,8,9,9-pentamethyl-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 26 June 2014 and 28 July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 03 October 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted July 2000

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese METI, MHLW and MOE Guidelines (twenty-eight day repeat dose oral toxicity study)

Version / remarks:

adopted 21 November 2003 amended 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

7,7,8,9,9-pentamethyl-5H,6H,6aH,7H,8H,9H,9aH-cyclopenta[h]quinazoline

EC Number:

801-694-5

Cas Number:

1392325-86-8

Molecular formula:

C16H24N2

IUPAC Name:

7,7,8,9,9-pentamethyl-5H,6H,6aH,7H,8H,9H,9aH-cyclopenta[h]quinazoline

Test material form:

other: Solid

Details on test material:

Identification: IFF TM 12-206 (FRET 10-0199)
Description: White solid block
Storage conditions: Approximately 4 °C, in the dark

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Han™:RccHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately six to eight weeks old
- Weight at study initiation: males weighed 206 to 248 g, the females weighed 160 to 186 g
- Fasting period before study: Animals were not fasted prior to blood sampling and necropsy
- Housing: the animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding and environmental enrichment in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK)
- Diet: a powdered diet (Rat and Mouse SQC Ground Diet No. 1, Harlan Laboratories U.K. Ltd., Oxon, UK) was provided ad libitum
- Water: mains drinking water was supplied ad libitum from polycarbonate bottles attached to the cage
- Acclimation period: seven days

DETAILS OF FOOD AND WATER QUALITY:
The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): target range 22 ± 3 °C
- Humidity (%): target range 50 ± 20%
- Air changes (per hr): at least fifteen
- Photoperiod (hrs dark / hrs light): 12/12

The Study Plan target value for relative humidity controls was 50 ± 20%. The target range for relative humidity was exceeded on two days during the course of the study with the maximum humidity achieved being 73.69% RH. While it is accepted that these deviations from the target range for relative humidity was less than ideal, overall it was considered that these deviations had no adverse impact on the scientific purpose of the study.


IN-LIFE DATES: From: 12 September 2014 To: 24 October 2014

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
A known amount of test item was mixed with a small amount of basal laboratory diet until homogeneous in a Robot Coupe Blixer 4 set at a constant speed. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further thirty minutes at a constant speed, setting 1 in a Hobart QE 200 mixer.

DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures were prepared prior to the first treatment, and weekly thereafter.
- Storage temperature of food: The diet was stored in labelled, double plastic bags at approximately -18 °C and was fed daily.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by using a validated analytical procedure.

Samples were used for concentration analysis weekly. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration.
Triplicate sets of samples (side/middle/side) for all dose groups were used for homogeneity analysis. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
Stability analyses were performed for samples kept frozen for 3 weeks (500, 1000 and 20000 ppm and for samples keept frozen for 10 days (300 ppm) and the test item is found to be stable in the admixtures when the variation is <20% compared to the time-zero mean.

Duration of treatment / exposure:

The test item was administered continuously, for twenty-eight consecutive days, by dietary admixture. Control animals were treated in an identical manner with basal laboratory diet.
Recovery group animals were maintained for a further fourteen days treatment-free period following termination of treatment.

Frequency of treatment:

The test item was administered continuously by dietary admixture.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 other: ppm (22.1 mg/kg bw)

Doses / concentrations 2

Dose / conc.:

800 other: ppm (59.7 mg/kg bw)

Doses / concentrations 3

Dose / conc.:

1 200 other: ppm (87.4 mg/kg bw)

No. of animals per sex per dose:

Five animals / sex / dose.

Control animals:

yes, plain diet

other: control and high dose recovery groups included

Details on study design:

- Dose selection rationale: based on results of a dose-range finder (see supporting study Harlan Laboratories Ltd., 2014)
- Rationale for animal assignment: animals were randomly allocated to treatment groups using a stratified body weight randomization procedure
- Fasting period before blood sampling for clinical biochemistry: none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- Prior to the start of treatment and weekly thereafter, all animals were observed for signs of functional/behavioral toxicity. Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 (prior to the start of treatment) and at weekly intervals thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes except during Week 3 where water intake was measured gravimetrically

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of tretament (day 28) for all non-recovery test and control group animals and end of treatment-free period (day 42) for all recovery group animals
- Anaesthetic used for blood collection: Yes, suitable barbiturate was used (not further specified)
- Animals fasted: No
- Parameters examined: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC)), Total leukocyte count (WBC), Differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic). Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of tretament (day 28) for all non-recovery test and control group animals and end of treatment-free period (day 42) for all recovery group animals
- Anaesthetic used for blood collection: Yes, suitable barbiturate was used (not further specified)
- Animals fasted: No
- Parameters examined: Urea, Inorganic phosphorus (P), Glucose, Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin, Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+), Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Bile acids, Calcium (Ca++), Gamma glutamyltranspeptidase. Parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant.

URINALYSIS: Yes
- Time schedule for collection of urine: during week 4 for all non-recovery test and control group animals and during week 6 for all all recovery group animals.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, overnight (normal hydration)
- Parameters examined: Volume, Ketones, Specific Gravity, Bilirubin, pH, Urobilinogen, Protein, Blood, Glucose.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4 of the study
- Dose groups that were examined: all groups
- Battery of functions tested:
Functional Performance Tests
Motor Activity
Forelimb/Hindlimb Grip Strength was assessed using an automated grip strength meter

IMMUNOLOGY: No

THYROID HORMONE ASSESSMENT:
- Time schedule for collection of blood: At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored frozen at approximately -20 °C
- Parameters included: Triiodothyronine (T3), Thyroxine (T4) and Thyroxine Stimulating Hormone (TSH)

Sacrifice and pathology:

Pathology
Necropsy
On completion of the dosing period, or in the case of recovery group animals, at the end of the treatment-free period, all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation:
Adrenals, Liver, Brain, Ovaries, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Pituitary (post-fixation), Thyroid/Parathyroid (post fixation), Prostate and Seminal Vesicles (with coagulating glands and fluids), Uterus with Cervix.

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and pons), Rectum, Salivary glands (submaxillary), Caecum, Sciatic nerve, Colon, Seminal vesicles (with coagulating glands and fluids), Duodenum, Epididymides♦, Skin (hind limb), Esophagus, Spinal cord (cervical, mid thoracic and lumbar), Eyes*, Gross lesions, Spleen, Heart, Stomach, Ileum (including Peyer’s patches), Testes♦, Jejunum, Thymus, Kidneys, Thyroid/Parathyroid, Liver, Trachea, Lungs (with bronchi)#, Urinary bladder, Lymph nodes (mandibular and mesenteric), Uterus & Cervix, Mammary gland, Vagina, Muscle (skeletal).
* Eyes fixed in Davidson’s fluid
♦ Preserved in Modified Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

The tissues shown below from all non-recovery control and 1200 ppm dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed together with the liver and kidneys from all 300, 800 ppm and recovery dose group animals. In addition, sections of testes from all Control and 1200 ppm males were stained with Periodic Acid-Schiff (PAS) stain and examined.
Adrenals, Ovaries, Pituitary, Bone & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and pons), Rectum, Caecum, Sciatic nerve, Colon, Seminal vesicles (with coagulating glands and fluids), Duodenum, Epididymides, Spinal cord (cervical, mid thoracic and lumbar), Eyes, Gross lesions, Spleen, Heart, Stomach, Ileum (including Peyer’s patches), Testes, Jejunum, Thymus, Kidneys, Thyroid/Parathyroid, Liver, Trachea, Lungs (with bronchi), Urinary bladder, Lymph nodes (mandibular and mesenteric), Uterus & Cervix, Mammary gland, Vagina, Muscle (skeletal).

Since there were indications of treatment-related liver, kidney (males only), thyroid and stomach (females only) changes, examination was subsequently extended to include similarly prepared sections of the liver, kidney (males only), thyroids and stomach (females) from animals in the low, intermediate and recovery groups.

Statistics:

See other information on materials and methods

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs apparent during the study.

Mortality:

no mortality observed

Description (incidence):

There were no clinical signs apparent during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Females treated with 1200 ppm showed a statistically significant reduction in body weight gain during the first two weeks of treatment, with actual body weight losses evident during the first week. Females treated with 800 ppm also showed a reduction in body weight gain during Weeks 1 and 4 however statistical significance was not achieved. Although some recovery was evident in 1200 and 800 ppm females, overall body weight gains during the treatment period was lower than control females. During the treatment-free period however, body weight development for the high dose females was statistically significantly higher than controls during Week 5 of the study.

No toxicologically significant effects were detected in males treated with 1200 or 800 ppm or in animals of either sex treated with 300 ppm.

For recovery 1200 ppm males, a statistically significant reduction in body weight gain was evident during the final week of the treatment free period. In the absence of a similar effect in males during the treatment period the intergroup difference was considered not to be of toxicological significance.
Animals of either sex treated with 300 ppm showed a statistically significant reduction during Week 3 of treatment. In the absence of a true dose related response or an adverse effect on overall body weight gain at this level, the intergroup difference was considered to be of no toxicological importance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Females treated with 1200 ppm showed a reduction in food consumption during the first week of treatment. Recovery was evident thereafter.
No such effects on food consumption were detected in males treated with 1200 or 800 ppm, in animals of either sex treated with 300 ppm or in recovery animals during the treatment free period.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food conversion efficiency was reduced in females treated with 1200 ppm during the first two weeks of treatment. Females treated with 800 ppm also showed a reduction in food conversion efficiency during Weeks 1 and 4.
No such effects on food conversion efficiency were detected in males treated with 1200 or 800 ppm, in animals of either sex treated with 300 ppm or in recovery animals during the treatment free period.
Females treated with 300 ppm showed a reduction in food conversion efficiency during Week 3. No adverse effect on food consumption was evident at this level and the reduced food conversion efficiency was considered to be the result of the reduced body weight gain seen in these females during this week.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

No treatment-related adverse effects on water intake were observed.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the hematological parameters examined.
Females from all treatment groups showed statistically significant reductions in mean corpuscular hemoglobin and mean corpuscular volume and a statistically significant increase in platelet count. The majority of individual values were within the normal ranges for rats of the strain and age used and in the absence of a true dose related response in the case of platelet count, the intergroup differences were considered of no toxicological significance. Recovery 1200 ppm females showed statistically significant reductions in hemoglobin, hematocrit and erythrocyte count following fourteen days without treatment. In the absence of a similar effect detected in non-recovery animals at the end of the treatment period, the intergroup differences were considered to be of no toxicological significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, males treated with 1200 ppm showed increases in total protein, albumin, calcium concentration and a reduction in glucose. Males treated with 1200 and 800 ppm showed an increase in gamma glutamyltranspeptidase. Females treated with 1200 ppm showed increases in chloride and calcium concentration and a reduction in aspartate aminotransferase. Females from all treatment groups showed increases in total protein, gamma glutamyltranspeptidase and reductions in glucose and albumin/globulin ratio. Animals of either sex treated with 1200 and 800 ppm also showed an increase in cholesterol. The majority of female individual values for cholesterol and gamma glutamyl transpeptidase were outside of the normal expected ranges for these parameters. Although the majority of the individual values for all of the remaining intergroup differences were within the normal expected ranges and in the case of albumin/globulin ratio, a linear dose related response was not evident, the potential association with histopathological changes in the liver mean that a relationship to treatment cannot be excluded.
No toxicologically significant effects were detected in males treated with 300 ppm or in recovery animals following fourteen days without treatment.
Females treated with 300 ppm showed a statistically significant reduction in bilirubin whilst males from this treatment group showed a statistically significant increase in alanine aminotransferase. The majority of the individual values were within the normal expected range for rats of the strain and aged used and in the absence of a similar effect at 800 or 1200 ppm the intergroup differences were considered not to be of toxicological significance. Recovery 1200 ppm males showed a statistically significant reduction in aspartate aminotransferase following fourteen days without treatment. In the absence of a similar effect detected in non-recovery males at the end of the treatment period, the intergroup difference was considered to be of no toxicological significance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects detected in the urinalytical parameters examined.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no treatment-related changes in the behavioral parameters at 300, 800 and 1200 ppm.

There were no toxicologically significant changes in functional performance. Females treated with 1200 ppm showed a statistically significant reduction in the third test of fore limb grip strength. In the absence of a true dose related response or any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup difference was considered to be of no toxicological significance. Males treated with 800 ppm showed a statistically significant increase in the first test of fore limb grip strength whilst females from this treatment group showed a statistically significant increase in the first test of hind limb grip strength. In the absence of a true dose related response or any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered to be of no toxicological significance.

There were no treatment-related changes in sensory reactivity.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, animals of either sex treated with 1200 and 800 ppm and females treated with 300 ppm showed a statistically significant increase in liver weight both absolute and relative to terminal body weight. Males treated with 1200 ppm also showed a statistically significant increase in kidney weight both absolute and relative to terminal body weight.
No toxicologically significant effects were detected in males treated with 300 ppm or in recovery animals following fourteen days without treatment.
Females treated with 800 ppm showed a statistically significant increase in thymus weight both absolute and relative to terminal body weight. In the absence of a true dose related response or any associated histopathological correlates, the intergroup difference was considered of no toxicological importance. At the end of the treatment free period, recovery females showed a statistically significant reduction in absolute and relative adrenal weight and a statistically significant increase in absolute and relative heart weight. In the absence of a similar effect seen in non-recovery females following the cessation of treatment or any associated histopathological correlates, the intergroup differences were considered of no toxicological importance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic necropsy findings did not indicate any adverse effect of dietary exposure at 300, 800 or 1200 ppm.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: Centrilobular hypertrophy was evident in three males and one female treated with 300 ppm, in three males and all females treated with 800 ppm and in four males and all females treated with 1200 ppm. Partial regression was evident, with only two recovery males and two recovery females still showing the effect following fourteen days without treatment. Periportal cellular change (eosinophilic, homogenous cytoplasm) was evident in all males treated with 1200 and 800 ppm. It was not present in recovery animals following fourteen days without treatment. Periportal vacuolation (fat-type) was present in two males and all females treated with 800 ppm
and in all males and four females treated with 1200 ppm. It was present in only one recovery female following fourteen days without treatment.

Thyroids: Follicular epithelial hypertrophy was present in one male and two females treated with 300 ppm, in four males and all females treated with 800 ppm and in four males and all females treated with 1200 ppm. This persisted in only one recovery male following fourteen days without treatment.

Kidneys: An increase in incidence and severity of hyaline droplets was evident in all males from all treatment groups. Multifocal basophilic tubules was also present in one male treated with 300 ppm, in all males treated with 800 ppm and in four males treated with 1200 ppm. Both changes persisted in recovery males following fourteen days without treatment.

Stomach: Erosion or ulceration was present in the glandular region of two females treated with 1200 ppm. Inflammatory change, and/or non-glandular hyperplasia was present in two females treated with 800 ppm and in three females treated with 1200 ppm. Complete regression was evident in recovery females following fourteen days without treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone assessment: No treatment-related effects were observed on T3, T4 or TSH

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: increased liver weight accompanied by changes in clinical chemistry parameters and histopathological abnormalities

Remarks on result:

other:

Remarks:

Equivalent to 22.1 mg/kg bw/day for males and 22.6 mg/kg bw/day for females.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

800 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Results analytical verification of doses

The admixtures investigated during the study were found to comprise test item in the range of 93 % to 113 % and thus the required content limit of ± 20 % with reference to the nominal content was met. The test item was found to be stable in the admixtures when kept for 3 weeks frozen (84%, 96% and 96% of initial in 500, 1000 and 20000 ppm groups, respectively) and 10 days frozen (99% of intial for 300 ppm) due to results which met the variation limit of 20 % from the time zero mean.

Summary test substance related findings

		Males				Females
Dose level (ppm)		0	300	800	1200	0	300	800	1200
Test item intake	mg/kg bw/day	0	22.1	59.7	87.4	0	22.6	59.1	81.4
Terminal bodyweight		277	273.8	300.4	285.2	205.4	182.4	197.4	184.3
Total bw gain		67.9	49.8	72.2	61.1	33.7	16.4	23.4	10.8
% bw gain		30	22	32	27	19	10	13	6
Mean food consumption^a	Week 1-4 (g/animal/day)	19.8	18.8	20.1	18.8	16.0	13.4	14.0	11.9
Haematology
Platelets	10^9/L	678.2	735.0	691.6	790.8	598.0	756.2*	857.0**	847.8**
Clinical chemistry
yGT	IU/L	0.20	0.80	3.20**	6.60**	0.00	0.60*	5.80**	15.00**
Cholesterol	mg/dL	70.6	78.4	94.2**	102.6**	60.0	82.2	123.0**	187.6**
Total protein	g/dL	6.412	6.630	6.676	7.290**	6.278	7.188**	7.196**	7.756**
Organ weights
liver	g	8.83114	9.1158	12.4920**	12.4520**	7.4082	7.57056**	10.0773**	10.7410**
	rel. weight	3.186	3.33	4.16**	4.368**	3.593	4.152**	5.093**	5.872**
	% increase (rel. weight)		+5%	+31%	+37%		+16%	+42%	+63%
kidney	g	1.61988	1.7307	1.92384	1.90866*	1.15974	1.17772	1.26046	1.25578
	rel. weight	0.586	0.632	0.64	0.668*	0.568	0.647	0.645	0.687
	% increase (rel. weight)		+8%	+9%	+14%		+14%	+14%	+21%

^aMean of means of week 1-4

* p<0.05

** p<0.01

 

Summary findings histopathological examination

		Males				Females
Dose level	ppm	0	300	800	1200	0	300	800	1200
Liver	centrilobular hypertrophy	0/5	3/5	3/5	4/5	0/5	1/5	5/5	5/5
	periportal vacuolation	0/5	0/5	2/5	5/5	0/5	0/5	5/5	4/5
	periportal cellular change (eosinophilic, homogenous cytoplasm)	0/5	0/5	5/5	5/5	0/5	0/5	0/5	0/5
Kidney	hyaline droplets	1/5	5/5	5/5	5/5	0/5	-	-	0/5
	multifocal basophilic tubules	0/5	1/5	5/5	4/5	0/5	-	-	0/5
Thyroid	follicular epithelial hypertrophy	0/5	1/5	4/5	4/5	0/5	2/5	5/5	5/5
Stomach	Erosion glandular region	0/5	-	-	0/5	0/5	0/5	0/5	1/5
	ulceration glandular region	0/5	-	-	0/5	0/5	0/5	0/5	1/5
	Inflammatory change	0/5	-	-	0/5	0/5	0/5	1/5	1/5
	non-glandular hyperplasia	0/5	-	-	0/5	0/5	0/5	2/5	3/5

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test (OECD TG 407, GLP), the local NOAEL was determined to be 300 ppm corresponding to 22.1 mg/kg bw/day based on irritation effects in the stomach and the systemic NOAEL was determined to be 300 ppm corresponding to 22.1 mg/kg bw/day based on increased liver weight accompanied by changes in clinical chemistry parameters and histopathological abnormalities observed at 800 and 1200 ppm.

Executive summary:

In a subacute repeated dose toxicity study (OECD TG 407, GLP) the test substance was administered by continuous dietary admixture to three groups, each of five male and five female Han™:RccHan™:WIST strain rats at dietary concentrations of 300, 800 and 1200 ppm (equivalent to a mean achieved dosage of 22.1, 59.7, and 87.4 mg/kg bw/day respectively for males and 22.6, 59.1 and 81.4 mg/kg bw/day respectively for females). A control group of five males and five females were treated with basal laboratory diet. Two recovery groups, each of five males and five females, were treated with the high dose (1200 ppm) or basal laboratory diet for twenty-eight consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, body weight change, food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. All animals were subjected to gross necropsy examination and histopathological examination of selected tissues was performed.

Results, clinical signs: There were no unscheduled deaths. There were no clinical signs apparent and no treatment-related changes in behaviour functional performance tests or sensory reactivity test.

Females treated with 1200 ppm showed a reduction in body weight gain during the first two weeks of treatment, with actual body weight losses evident during the first week. Females treated with 800 ppm also showed a reduction in body weight gain during Weeks 1 and 4. Although some recovery was evident in 1200 and 800 ppm females, overall body weight gains during the treatment period were lower than control females but did not show a clear dose-response. Terminal bodyweight was reduced by 10% in females treated with 1200 ppm accompanied by a decrease in the body weight gain of 70% and was considered adverse. No toxicologically significant effects on bodyweight, bodyweight gain and food consumption were detected in males treated with 1200 or 800 ppm, in animals of either sex treated with 300 ppm or in recovery animals during the treatment free period. The reduced bodyweight gain in females was accompanied by lower food consumption of up to -25% in high dose females. No such effects were detected in males or in recovery animals during the treatment free period. No adverse effect on water consumption was observed. Overall, the effect on body weight (gain) and food consumption at 1200 ppm in females was considered to represent an adverse effect of treatment although reluctance to eat the dietary formulation at this dose level may have contributed to this effect. The NOAEL for effects on body weight (gain) is 1200 ppm / 81.4 mg/kg bw/day.

Haematology: There were no toxicologically significant effects detected in the haematological parameters examined.

Clinical chemistry: At the end of the treatment period, males treated with 1200 ppm showed increases in total protein, albumin, calcium concentration and a reduction in glucose. Males treated with 1200 and 800 ppm showed an increase in gamma glutamyltranspeptidase (yGT). Females treated with 1200 ppm showed increases in chloride and calcium concentration and a reduction in aspartate aminotransferase. Females from all treatment groups showed increases in total protein, yGT and reductions in glucose and albumin/globulin ratio. Animals of either sex treated with 1200 or 800 ppm also showed an increase in cholesterol. No such effects were detected in males treated with 300 ppm or in recovery animals following fourteen days without treatment.

There were no treatment-related effects detected in the urinalytical parameters examined.

Organs:  No treatment-related macroscopic abnormalities were detected.

Organ-liver: At the end of the treatment period, animals of either sex treated with substance showed an increase in liver weight both absolute and relative to terminal body weight. The increase in rel. liver weight were 5%, 31% and 38% and 16%, 42% and 63% at 300 ppm, 800 ppm and 1200 ppm in males and females, respectively. Centrilobular hypertrophy was evident in animals of either sex from all treatment groups. Periportal vacuolation (fat-type) was evident in animals of either sex treated with 1200 or 800 ppm. Periportal cellular change (eosinophilic, homogenous cytoplasm) was also evident in males treated with 1200 or 800 ppm. All of these changes showed evidence of reversibility in the recovery period within the study.

The effects observed at the mid and the high dose, increases in liver weight accompanied by changes in liver-related clinical chemistry parameters, such as yGT, total protein and cholesterol and histopathological abnormalities, were considered adverse. Effects observed at the low dose level (rel. weight < 16% and hypertrophy in females in 3/5 animals) are considered adaptive. Therefore the NOAEL for liver effects is set at 300 ppm / 22.1 mg/kg bw/day.

Organ-kidney effects: At the end of the treatment period, males and females treated with 1200 ppm showed an increase in kidney weight both absolute and relative to terminal body weight (not statistically significant in females). Increased hyaline droplets and multifocal basophilic tubules were evident in males from all treatment groups. These findings are linked to the accumulation of alpha 2u-globulin, which is unique to the male rat and considered to be of no relevance to man and therefore not considered relevant for NOAEL derivation.

Organ-Thyroid: Follicular epithelial hypertrophy was evident in animals of either sex from all treatment groups. This finding persisted in one recovery male only following the recovery period. These effects can be the result of hepatocellular induction and enhanced hepatic metabolism. Thyroid hormone levels were unaffected by treatment.

Organ-Stomach: Irritation effects of the substance such as erosion or ulceration were present in the glandular region the stomach of females treated with 1200 ppm. Inflammatory change, and/or non-glandular hyperplasia was also present in females treated with 800 and 1200 ppm. Complete regression was evident in recovery females following fourteen days without treatment.

Conclusion: Oral administration of the substance to rats for a period of twenty-eight consecutive days at dietary concentrations of 300, 800 and 1200 ppm resulted in treatment-related effects detected in animals of either sex from all treatment groups. The effect on body weight (gain) and food consumption at 1200 ppm was considered to represent an adverse effect of treatment. Local irritation effects were observed in the stomach at the mid and high dose levels with a NOAEL at 300 ppm / 22.1 mg/kg bw/day. The liver is considered a target organ and relative increases in liver weight and effects including clinical chemistry and histopathological changes were observed from the low dose onwards. The liver shows a dose related increase in processing the substance resulting in hypertrophy (at the low dose), periportal vacuolation and eosinophilic cytoplasm at the mid and high dose levels. It seems that from the mid dose onwards the liver becomes overloaded. The function of the liver was not impaired but the effects are considered adverse. Therefore the NOAEL is 300 ppm / 22.1 mg/kg bw/day. The NOAEL for local effects is 300 ppm equivalent to 22.1 mg/kg bw/day based on irritation effects in the stomach and the NOAEL for systemic effects is 300 ppm equivalent to 22.1 mg/kg bw/day based on the liver effects at higher doses.